Obesity is an increasingly prevalent global disease and has reached epidemic proportions. Current estimates suggest that at least 50% of the Western population is either overweight or obese. Obesity, particularly abdominal obesity, combined with other conditions such as insulin resistance, dyslipidemia, hepatic steatosis, and hypertension is known as the Metabolic, or Insulin Resistance, Syndrome. The central pathophysiological features of the dyslipidemia associated with insulin resistance and type 2 diabetes are increased plasma triglycerides (TG) in very low density lipoproteins (VLDL), and reduced high density lipoprotein (HDL) cholesterol. Commonly, increased circulating TGs are hydrolyzed into free fatty acids (FFA) and are taken up by peripheral tissues including the liver and can lead to hepatic steatosis, or non-alcoholic fatty liver. Studies of several mutant mouse models of obesity and metabolic disorders suggest that the link between insulin resistance and dysregulated TG is complex and involves both peripheral and central factors.
Insulin resistance refers to reduced insulin-stimulated glucose uptake in skeletal muscle and fat, and an impaired suppression of liver glucose output (2). Hyperglycemia and hyperlipidemia are both side effects of, and causative agents in, the pathophysiology of type 2 diabetes. Glucotoxicity and lipotoxicity further promote insulin resistance and type 2 diabetes due to suppression of insulin action and secretion from the β-cell. Hyperinsulinemia is initially successful in suppressing liver glucose output, however the deleterious effects of increased insulin offset the gains associated with maintaining normal blood glucose levels (2). Hyperinsulinemia is thought to be a factor in a cluster of metabolic abnormalities, including hypertension, non-alcoholic fatty liver disease (NAFLD) and coronary heart disease (2). NAFLD disease is commonly associated with insulin resistance, and requires two transcription factors: sterol regulatory element binding protein-1c (SREBP1c) and peroxisome proliferator receptor-γ (PPARγ) (3-6). Absence of SREBP1, or PPARγ signaling in liver inhibits the development of liver steatosis that occurs in obese insulin resistant mice (5-7).
Defining a common mechanism explaining insulin resistance has been difficult because of the complexity of the insulin receptor (IR) signaling system, and the realization that it is not one, but many factors that contribute to the development of this disorder. The tyrosine phosphorylation of two adaptor proteins, IRS1 and IRS2, is a critical early step in the stimulation of glucose uptake by insulin (8-11). IRS1 and IRS2 have no intrinsic enyzmatic activity, and are thought to function as part of a molecular scaffold that facilitates the formation of complexes of proteins with kinase, phosphatase or ubiquitin ligase function (12). Stimulation of phosphoinositide 3′ kinase (PI3K) by association with the IRS is a critical step in insulin-stimulated glucose uptake. Activation of the p110 catalytic subunit of PI3K activates the lipid kinase domain, which phosphorylates phosphatidylinositol-4,5-bisphosphate. Activation of PI3K is necessary for full stimulation of glucose uptake by insulin, although other pathways might also be involved (12).
A metabolic state conducive to the development of insulin resistance is thought to result from an imbalance of caloric intake with oxidative metabolism (13,14). Studies suggest that reduced mitochondrial function in muscle is a factor in the development of insulin resistance associated with obesity (14,15). Stimulation of energy expenditure and suppression of appetite both result in improved glucose metabolism in mouse models of obesity and type 2 diabetes. A well-characterized example of this is the adipocytokine leptin. Leptin acts in the hypothalamus and hindbrain to suppress appetite and through stimulation of the autonomic nervous system increases oxidative metabolism in skeletal muscle (16-20). However, leptin can also improve hepatic insulin sensitivity independently of marked effects on food intake or body weight (17).
Infusion of fatty acids (FA) is associated with rapid reductions in insulin sensitivity in muscle within 4-6 h (21-23). The exact mechanism by which FA's reduce insulin-stimulated glucose uptake remains a matter of debate. Recent data indicate that FA's interfere with the IR signal transduction pathways that stimulate glucose uptake (21,22,24). One hypothesis is that an increase in the intracellular concentration of FA's and diacyl-glycerol leads to the activation of a serine kinase, protein-kinase C θ (PKCθ) (25). Phosphorylation of IRS1 on Ser307 by PKCθ inhibits the phosphorylation of IRS-1 by the IR, leading to reduced activation of PI3K and a reduction in the stimulation of glucose uptake by insulin.
Abnormal Activity of Secreted Polypeptides as a Link Between Obesity and Insulin Resistance.
Determining mechanisms linking obesity with insulin resistance is important for developing new glucose lowering therapies. Recent research investigating insulin resistance has focused on adipocytes. Obesity is associated with aberrant regulation and function of a regulatory network of polypeptides secreted from adipocytes (adipocytokines). Adipocytokines such as leptin, adiponectin, and resistin regulate hepatic glucose production, glucose disposal in muscle, and the proliferation and storage of lipid in adipocytes (26). Leptin regulates energy homeostasis through effects on neurons located in the hypothalamus and hindbrain, regulating ingestive behavior, autonomic nervous activity, and neuroendocrine system that govern metabolism (thyroid, adrenals) (16). Leptin resistance or reduced serum adiponectin associated with obesity are factors that contribute to insulin resistance, through diminished insulin-sensitizing actions and by increasing risk for developing steatosis (intracellular fatty acid accumulation) (27,28). Non-adipose tissues also secrete peptides that affect energy metabolism and insulin sensitivity, such as musclin from muscle (29) and angiopoietin-related growth factor from liver (30). These factors may also be targets for the treatment of the metabolic syndrome.
Melanocortin Receptor Knockouts for Investigating the Link Between Obesity and Insulin Resistance:
Two melanocortin receptors expressed in areas of the central nervous system are involved in energy homeostasis. Targeted deletion of the neuronal melanocortin-4 receptor (MC4R) gene in mice (Mc4r−/− or Mc4rKO mice) causes obesity and hyperinsulinemia, and is also associated with increased hepatic lipogenic gene expression and hepatic steatosis. Mice deficient for another neuronal melanocortin receptor (Mc3r−/− or Mc3rKO mice) develop a similar degree of obesity to Mc4r−/− mice when fed a high fat diet, but do not exhibit the same level of insulin resistance, hyperlipidemia and increased hepatic steatosisWork Mc3rKO and Mc4rKO on the C57BL/6J (B6) strain both exhibit an exaggerated diet-induced obesity, however the deterioration of insulin sensitivity in Mc4rKO is more rapid and severe (31,32). FIGS. 1A-1E illustrate some of the known differences in wild-type mice (C57BL/6J) and the two knockout mice in terms of body mass as a function of either a low fat diet or a high fat diet. (31,32) Severe insulin resistance in mice and humans is associated with hepatomegaly and steatosis, with increased hepatic lipogenesis (33). Mc4rKO develop hepatic insulin resistance and hepatomegaly in the obese state, and on a high fat diet (HFD) exhibit a marked deterioration of glucose homeostasis associated with severe glucose and insulin intolerance. FIGS. 2A-2E show the differences in hepatomegaly and steatosis in the two mouse strains, and also differences in expression of genes involved in lipid metabolism. (4,17,57) On the other hand, Mc3rKO matched to Mc4rKO for fat mass (FM) exhibit a very modest impairment of glucose homeostasis.
Sequences of cDNA Similar to Enho1.
A sequence and putative open reading frame of a cDNA encoding a putative protein homologous to ENHO1 have previously been published by several consortiums involved in large-scale sequencing of cDNAs. See R. L. Stausberg et al., “Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences,” Proc. Natl. Acad. Sci. U.S.A., vol. 99, pp. 16899-16903 (2002) (Genbank accession number: BC021944, cDNA with complete coding sequence); and H. F. Clark et al., “The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment,” Genome Res., vol. 13, pp. 2265-2270 (Genbank accession number: NM—198573, cDNA with complete coding sequence). A protein with similar homology for the first 37 amino acid residues of SEQ ID NO:2 has been identified. However, the nucleotide and amino acid sequence of this described protein may be incorrect, due to a single nucleotide error in the sequencing of the cDNA.